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  Method and composition for inhibiting or slowing blood coagulationUSPTO Application #: 20060035816Title: Method and composition for inhibiting or slowing blood coagulation Abstract: A method and composition for inhibiting or slowing blood coagulation includes lactadherin, a fragment of lactadherin, a functional equivalent of lactadherin, or a functional equivalent of a fragment of lactadherin. (end of abstract)
Agent: Dinesh Agarwal Law Office Dinesh Agarwal - Alexandria, VA, US Inventors: Gary E Gilbert, Jialin Shi USPTO Applicaton #: 20060035816 - Class: 514008000 (USPTO) Related Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), Peptide Containing (e.g., Protein, Peptones, Fibrinogen, Etc.) Doai, Glycoprotein (carbohydrate Containing) The Patent Description & Claims data below is from USPTO Patent Application 20060035816. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application claims priority on prior U.S. Provisional Application Ser. No. 60/386,562, filed Jun. 7, 2002, and which is incorporated herein in its entirety by reference. BACKGROUND OF THE INVENTION [0003] The present invention is generally directed to inhibiting or slowing blood coagulation, and more particularly to using lactadherin or a fragment thereof as an agent for inhibiting or slowing blood coagulation. [0004] There are several anticoagulant drugs which are in widespread clinical use and many others in development, including clinical trials. However, none of these agents have the mechanism of blocking access of blood proteins to procoagulant membrane surfaces. Inhibition of coagulation at this stage is an earlier step than most anti-coagulants target and, it is specifically an anticoagulant step as opposed to a step which would inhibit platelet aggregation or adhesion. [0005] Investigators have sought anticoagulants that would work via this mechanism for decades. Phospholipases from snake venoms can function as anticoagulants by competing for membrane binding sites and the function of several phospholipases have been analyzed in detail. These proteins are unsuitable for use in humans because the corresponding enzymatic activity (phospholipases cleave phospholipids releasing lysophospholipids and free fatty acids) cause inflammation and tissue injury. A single class of agents, annexins, have been shown to inhibit blood coagulation by blocking the membrane surface. These proteins have been evaluated as anticoagulants in animals. The efficacy is modest, at least partly because annexin is more fastidious in its requirements for membrane lipids than most blood clotting proteins. Thus, much of the procoagulant membrane of cells remains unblocked, even with a vast excess of annexin V. We compared the anticoagulant efficacy of annexin V directly to that of lactadherin. [0006] Lactadherin is a MW 47,000 glycoprotein of milk fat globules. It has also been known as PAS-6/7, indicating the two glycosylation variants (Reference 1), bovine-associated mucoprotein, BA-46, P47, and MFG-E8 (Reference 2). Lactadherin has a domain structure of EGF1-EGF2-C1-C2 in which EGF indicates epidermal growth factor homology domains, and the C domains share homology with the discoidin family including the lipid-binding "C" domains of blood coagulation factor VIII and factor V (FIG. 1) (Reference 2). The second EGF domain displays an Arg-Gly-Asp motif (Reference 3) which binds to the .alpha..sub.v.beta..sub.5 and .alpha..sub.v.beta..sub.5 integrins (References 1 and 4-6). The second C domain binds to phospholipids (Reference 6). [0007] In milk fat globules, lactadherin lines the surface of the phospholipid bilayer which surrounds the central triglyceride droplet, apparently stabilizing the bilayer (Reference 7). Lactadherin decreases the symptoms of rotavirus infection in infants, possibly by binding to rotavirus particles via carbohydrate moieties (Reference 8). In tissue sections, lactadherin is found localized on the apical portion of secretory epithelium in the breast (Reference 7). Abundant expression by breast carcinoma tissue makes lactadherin a potential target for antigen-guided radiation therapy (Reference 9). Lactadherin is also found on the apical surface of epithelia in the biliary tree, the pancreas, and sweat glands (Reference 7) and is synthesized by aortic medial smooth muscle cells (Reference 10). Function in these tissues remains unknown. Lactadherin has been identified as a zona pellucida-binding protein on the acrosomal cap of sperm (Reference 11). [0008] Blood coagulation factor VIII and factor V bind to phospholipid membranes via "C" domains which share homology with lactadherin (References 12-14). Remarkable features of membrane binding for these proteins include high affinity (K.sub.D approx. 2 nM) (Reference 15) and sufficient specificity so that no plasma proteins compete for membrane binding sites (Reference 16). Factor VIII binds via stereo-selective interaction with the phospho-.sub.L-serine motif of phosphatidylserine (PS) (Reference 17). Factor V also exhibits stereoselective interaction with PS (Reference 18). Binding of factor VIII is enhanced by the presence of phosphatidylethanolamine (PE) in the membrane (Reference 19), by unsaturated phospholipid acyl chains (Reference 20), and by membrane curvature (Reference 19). The crystal structures of the C2 domains of factors VIII and V suggest that membrane binding is mediated by two pairs of hydrophobic residues displayed at the tips of .beta.-hairpin turns (References 21-22). Mutagenesis studies have confirmed the role of these residues in phospholipid binding (Reference 23). The homology of the lactadherin C domains with those of factors VIII and V suggests that similar phospholipid binding properties may exist. Indeed, lactadherin has been found to bind selectively to PS (Reference 24) and to utilize primarily the C2 domain in its lipid binding (Reference 6). [0009] Annexin V, like factor VIII and factor V, exhibits high affinity, PS-dependent membrane binding (Reference 25). However, the quadruplicate membrane-binding motifs of annexin V are not homologous with the discoidin-like domains of lactadherin, factor VIII, and factor V (Reference 26). Corresponding to the difference in structure, the membrane binding mechanism is different. In addition, annexin V requires Ca.sup.++ for membrane-binding and the binding is chiefly hydrophilic in nature (Reference 27). Annexin V does have the capacity to compete for a fraction of the phospholipid binding sites utilized by the factor VIIIa-factor IXa enzyme complex and the factor Xa-factor Va enzyme complex of the coagulation cascade so that it functions in vitro as a membrane-blocking anticoagulant (Reference 28). The well-defined membrane-binding and anti-coagulant properties of annexin V make studies with annexin V suitable controls for studies with lactadherin. OBJECTS AND SUMMARY OF THE INVENTION [0010] The principal object of the present invention is to provide an agent which inhibits or slows blood coagulation. The agent includes lactadherin, a fragment of lactadherin, a functional equivalent of lactadherin, or a functional equivalent of a fragment of lactadherin. [0011] An object of the present invention is to provide an agent which inhibits or slows blood coagulation by competing for membrane binding sites of factor VII and/or factor V. [0012] Another object of the present invention is to provide an agent which inhibits or slows blood coagulation by inhibiting the prothrombinase complex and the factor VII a-tissue factor complex. [0013] Yet another object of the present invention is to provide an agent which inhibits or slows clotting of whole blood. [0014] Still yet another object of the present invention is to provide an agent which inhibits or slows coagulation reactions on cell membranes. [0015] An additional object of the present invention is to provide an agent which is an efficient inhibitor of the prothrombinase and the factor Xase complex regardless of the degree of membrane curvature and/or the phosphatidylserine content. [0016] Yet an additional object of the present invention is to provide an agent which is more effective than annexin V in inhibiting or slowing blood coagulation or clotting. [0017] Lactadherin, a glycoprotein of the milk fat globule membrane, contains tandem C-domains with homology to discoidin-type lectins and to membrane-binding domains of blood-clotting factors V and VIII. We investigated whether the structural homology confers the capacity to compete for the membrane-binding sites of factor VIII and factor V and to function as an anticoagulant. Our results indicate that lactadherin competes efficiently with factor VIII and factor V for binding sites on synthetic phosphatidylserine-containing membranes with half-maximal displacement at lactadherin concentrations of 1-4 nM. Binding competition correlated to functional inhibition of factor VIIIa-factor IXa (factor Xase) enzyme complex. In contrast to annexin V, lactadherin was an efficient inhibitor of the prothrombinase and the factor Xase complexes regardless of the degree of membrane curvature and the phosphatidylserine content. Lactadherin also inhibited the factor Vila-tissue factor complex efficiently whereas annexin V was less effective. Since the inhibitory concentration of lactadherin was proportional to the phospholipid concentration, and because lactadherin was not an efficient inhibitor in the absence of phospholipid, the major inhibitor effect of lactadherin relates to blocking phospholipid sites rather than forming inhibitory protein-protein complexes. Lactadherin was also found to be an effective inhibitor of a modified whole blood prothrombin time assay in which clotting was initiated by dilute tissue factor; 60 nM lactadherin prolonged the prothrombin time 150% vs. 20% for 60 nM annexin V. These results indicate that lactadherin functions as a potent phospholipid-blocking anticoagulant. [0018] At least one of the above-noted objects is met, in part, by the present invention, which in one aspect includes blocking or reducing access to a procoagulant molecule by a coagulation molecule by subjecting a procoagulant molecule to lactadherin, a fragment of lactadherin, a functional equivalent of lactadherin, or a functional equivalent of a fragment of lactadherin. [0019] Another aspect of the invention includes blocking or reducing binding of a ligand to cell membrane binding site by subjecting a cell membrane binding site to lactadherin, a fragment of lactadherin, a functional equivalent of lactadherin, or a functional equivalent of a fragment of lactadherin. [0020] Another aspect of the invention includes inhibiting or slowing blood coagulation by subjecting a predetermined amount of blood to an effective amount of lactadherin, a fragment of lactadherin, a functional equivalent of lactadherin, or a functional equivalent of a fragment of lactadherin. [0021] Another aspect of the invention includes inhibiting or slowing blood clotting by administering to a subject in need thereof an effective amount of lactadherin, a fragment of lactadherin, a functional equivalent of lactadherin, or a functional equivalent of a fragment of lactadherin. [0022] Another aspect of the invention includes preventing or reducing inflammation by administering to a subject in need thereof an effective amount of lactadherin, a fragment of lactadherin, a functional equivalent of lactadherin, or a functional equivalent of a fragment of lactadherin. Continue reading... Full patent description for Method and composition for inhibiting or slowing blood coagulation Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Method and composition for inhibiting or slowing blood coagulation patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. 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